A. Histocompatibility and Transplant Rejection:
Histocompatibility is a largely unsolved problem in transplant medicine. Rejection of transplanted tissue is the result of an adaptive immune response to alloantigens on the grafted tissue by the transplant recipient. The alloantigens are “non-self proteins, i.e., antigenic proteins that vary among individuals in the population and are identified as foreign by the immune system of a transplant recipient. The antigens on the surfaces of transplanted tissue that most strongly evoke rejection are the blood group (ABO) antigens and the major histocompatibity complex (MHC) proteins and in the case of humans, the human leukocyte antigen (HLA) proteins.
The blood group antigens were first described by Landsteiner in 1900; they are branched oligosaccharides that are attached to proteins and lipids on the surfaces of red blood cells, endothelial cells, and other cells, and are also present in secretions such as saliva. Compatibility of the blood group antigens of the ABO system of a vascularized organ or tissue transplant with those of the transplant recipient is generally required; but blood group compatibility may be unnecessary for many types of cell transplants.
The HLA proteins are encoded by clusters of genes that form a region located on chromosome 6 known as the Major Histocompatibility Complex, or MHC, in recognition of the important role of the proteins encoded by the MHC loci in graft rejection. Accordingly, the HLA proteins are also referred to as MHC proteins. The MHC genes and proteins will be used interchangeably in this application as the application encompasses human and non-human animal applications. The HLA or MHC proteins normally play a role in defending the body against foreign pathogens such as viruses, bacteria, and toxins. They are cell surface glycoproteins that bind peptides at intracellular locations and deliver them to the cell surface, where the combined ligand is recognized by a T cell. Class I MHC proteins are found on virtually all of the nucleated cells of the body. The class I MHC proteins bind peptides present in the cytosol and form peptide-MHC protein complexes that are presented at the cell surface, where they are recognized by cytotoxic CD8+ T cells. Class II MHC proteins are usually found only on antigen-presenting cells such as B lymphocytes, macrophages, and dendritic cells. The class II MHC proteins bind peptides present in a cell's vesicular system and form peptide-MHC protein complexes that are presented at the cell surface, where they are recognized by CD4+ T cells. CD4+ T cells activated by class II MHC proteins undergo clonal expansion with production of regulatory cytokines that signal helper and cytotoxic T cells. Unfortunately for those in need of transplants, the frequency of T cells in the body that are specific for non-self MHC molecules is relatively high, with the result that differences at MHC loci are the most potent critical elicitors of rejection of initial grafts. Rejection of most transplanted tissues is triggered predominantly by the recognition of class I MHC proteins as non-self proteins. T cell recognition of foreign antigens on the transplanted tissue sets in motion a chain of signaling and regulatory events that causes the activation and recruitment of additional T cells and other cytotoxic cells, and culminates in the destruction of the transplanted tissue. (Charles A. Janeway et al., Immunobiology, Garland Publishing, New York, N.Y., 2001, p. 524).
B. The Genes Encoding MHC Proteins:
The MHC genes are polygenic—each individual possesses multiple, different MHC class I and MHC class II genes. The MHC genes are also polymorphic—many variants of each gene are present in the human and non-human population. In fact, the MHC genes are the most polymorphic genes known. Each MHC Class I receptor consists of a variable α chain and a relatively conserved β2-microglobulin chain. Three different, highly polymorphic class I α chain genes have been identified. These are called HLA-A, HLA-B, and HLA-C. Variations in the α chain chains account for all of the different class I MHC genes in the population. MHC Class II receptors are also made up of two polypeptide chains, an α chain and a β chain, both of which are polymorphic. In humans, there are three pairs of MHC class II α and β chain genes, called HLA-DR. HLA-DP, and HLA-DQ. Frequently, the HLA-DR cluster contains an extra gene encoding a β chain that can combine with the DR α chain; thus, an individual's three MHC Class II genes can give rise to four different MHC Class II molecules.
In humans, the genes encoding the MHC class I α chains and the MHC class II α and β chain are clustered on the short arm of chromosome 6 in a region that extends over from 4 to 7 million base pairs that is called the major histocompatibility complex. Every person usually inherits a copy of each HLA gene from each parent. If an individual's two alleles for a particular MHC locus encode structurally different proteins, the individual is heterozygous for that MHC allele. If an individual has two MHC alleles that encode the same MHC molecule, the individual is homozygous for that MHC allele. Because there are so many different variants of the MHC alleles in the population, most people have heterozygous MHC alleles. The numbers of different alleles found for each type of MHC class I α chain and MHC class II α and β chains as of January 2003 are shown in Table 1.
TABLE 1The numbers of different alleles for the polymorphic MHC classI and class II chains identified as of January, 2003.MHC chainno. of allelesHLA-A266HLA-B511HLA-C6HLA-DRA3HLA-DRB403HLA-DQA123HLA-DQB153HLA-DPA120HLA-DPB1101
The data in Table 1 is from the Internet web site of the Informatics Group of the Anthony Nolan Trust, The Royal Free Hospital, Hampstead, London, England. Lists of identified HLA Class I and Class II alleles are also available at the same web site.
C. Matching MHC Types to Inhibit Rejection of Transplants:
Since the recognition that-non-self-M1-G-molecules are a major determinant of graft rejection, much effort has been put into developing assays to identify the MHC types present on the cells of tissue to be transplanted, and on the cells of transplant recipients, in order to match the types of MHC molecules present in the transplant tissue with those of the recipient. Tissue typing, the detection of MHC antigens, is performed by various means; for example, (i) by serology, using antibodies specific for particular MHC molecules to detect the presence of the targeted MHC molecules on donor or recipient cells, e.g., by the lymphocytotoxicity test; (ii) by detection of antibodies of a transplant recipient that bind specifically to a MHC protein of transplant tissue; and (iii) by direct analysis of the nucleotide sequence of the DNA of the MHC alleles. Most tissue typing for organ banking purposes is done by determining the blood type (ABO typing) and by typing the patient's and donor cells using serological methods; however, the use of rapid and reliable DNA-specific methods is increasing. Such methods can employ sequence-specific oligonucleotide primers and amplification by the polymerase chain reaction (PCR), and can be augmented by combining fluorescent detection methods with the use of a DNA chip to which are bound sequence specific oligonucleotides designed to detect unique sequences present in the different MHC alleles.
At present, tissue typing to match the HLA antigens of a transplant with those of a recipient is usually limited to the Class I HLA-A and -B antigens, and the Class II HLA-DR antigens. Most transplant donors are unrelated to the transplant recipient, and finding a tissue type to match that of the recipient usually involves matching the blood type and as many as possible of the 6 HLA alleles—two for each HLA-A, -B, and -DR locus. Transplant centers do not usually consider potential incompatibilites at other FILA loci, such as HLA-C and HLA-DPB1, although mismatches at these loci can also contribute to rejection. Considering only the combinations of maternal and paternal alleles of the HLA-A. HLA-B, and HLA-DR loci identified to date, there is a complexity of billions of possible tissue types. The task of matching HLA types of organs for transplant is simplified in that HLA-A and HLA-B are usually identified serologically. The number of HLA antigens identified serologically is considerably less than the number of different MLA antigens based on DNA sequencing. The World Health Organization (WHO) has recognized 28 distinct antigens in the HLA-A locus and 59 in the HLA-B locus, based on serological typing. Matching organs is also simplified to some extent by the fact that some alleles are much more common than others. Some of the more common HLA-A and HLA-B alleles are shown in Table 2:
TABLE 2Frequency of common HLA-A and HLA-Balleles in the population.HLA-A (Frequency (%))HLA-A1(25.1)HLA-A2(44.8)HLA-A3(22.6)HLA-A24(18.2)HLA-A11(11.8)HLA-A28(9.8)HLA-A29(10.3)HLA-A32(9.8)HLA-B15(12.3)HLA-B (Frequency (%))HLAB5(15.2)HLA-B7(18.2)HLA-B8(16.7)HLA-B12(32.5)HLAB14(8.8)HLA-B18(11.3)HLA-B35(15.2)HLA-B40(13.7)(from Snell GD et al, Histocompatibility, New York, Academic Press, 1976)
The frequencies with which the various alleles appear in a population is not random; it depends on the racial makeup of the population. Dr. Motomi Mori has determined the frequencies with which thousand of different haplotypes of HLA-A, -B, and -DR loci appear in Caucasian, African-American, Asian-American, and Native American populations. Each haplotype is a particular combination of HLA-A, HLA-B, and HLA-DR loci that is present on a single copy of chromosome no. 6. The frequencies of several relatively common HLA-A, -B, and -DR haplotypes are shown in Table 3 to illustrate the wide variation in HLA haplotype frequencies in some of the racial groups that make up the North American population. In interpreting haplotype frequency data such as that shown in Table 3, one must bear in mind that cells of patients and organs are diploid and have an HLA type that is the product of the HLA haplotypes of the chromosomes inherited from both parents.
TABLE 3Examples of HLA-A, -B, -DR haplotype frequenciesHLA-A, -B, and -DR haplotype frequencies (expressed in percent) andtheir respective rankings within each racial group: Caucasian (CAU),African-American (AFR), Asian-American (ASI) and Native American (NAT).HaplotypeFrequency (%)RankingABDRCAUafrASILATNATCAUAFRASILATNAT1720.53490.20940.07980.18880.2812215826291621835.18121.24910.31951.67334.743912543121410.15630.04440.00760.37940.062410753914513931223540.14570.07370.32931.28580.63421153024941223580.08230.09310.17561.76410.328924122612214624442.15070.65060.12760.69062.0004341701233722.62850.75960.18911.19862.708323113523740.44110.15340.04980.17950.44483010440898293780.08480.03670.00000.06220.05372306531405331036633511.02240.27410.13720.35520.8125729156448315140.09150.03420.16460.25970.56912096991356416321470.26170.05130.00460.13240.1775574791858140104
The data in Table 3 was produced for The National Marrow Donor Program Donor Registry, and is available at the Internet web site of Motomi Mori, Ph.D., Huntsman Cancer Institute, Salt Lake City, Utah.
D. Rejection Triggered by Minor Histocompatibility Antigens:
Matching the MHC molecules of a transplant to those of the recipient significantly improves the success rate of clinical transplantation; however, it does not prevent rejection, even when the transplant is between HLA-identical siblings. This is because rejection is also triggered by differences between the minor histocompatibility antigens. These polymorphic antigens are actually “non-self peptides bound to MHC molecules on the cells of the transplant tissue. The rejection response evoked by a single minor histocompatibility antigen is much weaker than that evoked by differences in MHC antigens, because the frequency of the responding T cells is much lower (Janeway et al., supra, page 525). Nonetheless, differences between minor histocompatibility antigens will often cause the immune system of a transplant recipient to eventually reject a transplant, even where there is a match between the MHC antigens, unless immunosuppressive drugs are used.
E. Inadequate Supply of Cells, Tissues, and Organs for Transplant.
The number of people in need of cell, tissue, and organ transplants is far greater than the available supply of cells, tissues, and organs suitable for transplantation. Under these circumstances, it is not surprising that obtaining a good match between the MHC proteins of a recipient and those of the transplant is frequently impossible, and many transplant recipients must wait for an MHC-matched transplant to become available, or accept a transplant that is not MHC-matched. If the latter is necessary, the transplant recipient must rely on heavier doses of immunosuppressive drugs and face a greater risk of rejection than would be the case if MHC matching had been possible. There is presently a great need for new sources of cells, tissues, and organs suitable for transplantation that are histocompatible with the patients in need of such transplants.